Retroviral vector hybrids and the use thereof for gene transfer

ABSTRACT

Replication-defective retroviral vector hybrids which are characterized in that they contain a) the U5 region and/or tRNA primer binding site of MESV as the U5 region and/or tRNA primer binding site in the leader region and b) the U3 and R regions from a Friend murine leukaemia virus (F-MuLV) as the U3 and R regions in the 3&#39;-LTR and which are particularly suitable for an effective gene transfer in haematopoietic stem cells.

This application is a 371 of PCT/EP95/03175, filed Aug. 10, 1995.

The invention concerns retroviral vector hybrids and their use for gene transfer in particular for gene transfer into haematopoietic stem cells (ES).

Retroviral vectors with replication defects are at present a standard in gene transfer and gene therapy applications on cells of the human blood-forming system (A. D. Miller (1992) (1), R. C. Mulligan (1993) (2), R. Vile and S. J. Russell (1994) (3)). Their main advantages are:

the infection leads to a stable integration of the vector and of the DNA sequences transferred by it into the host genome of mitotically active cells

the number of integrations into the host genome can be controlled via the infection conditions

the safety aspects of retroviral gene transfer are well researched; complications have so far not occurred in numerous applications on humans.

The most frequently cited advantage of retroviral vectors, their high gene transfer efficiency, only partly applies to applications on the blood-forming system. Only late maturing blood-forming precursor cells can preferably be infected with conventional vectors based on the Moloney murine sarcoma virus (MoMuSV); 30-95% of the precursor cells transduced with these vectors exhibit either no or an inadequate expression of the transferred genes due to primary silencing mechanisms (4); moreover after a longer residence period in vivo the retrovirally controlled gene expression is weakened in a high percentage of the initially expressing cells up to the point of non-function due to secondary silencing (Palmer et al. (1991) (5), Brenner et al. (1993) (6)).

It has been demonstrated that also in embryonic cells such as embryonic carcinoma cells (myeloid (non-lymphatic) derivatives of haematopoietic stem cells) the viruses are indeed integrated but the expression of the integrated provirus is blocked.

Mutations and derivatives of MoMuSV are known from Stocking et al. (1993) (21) which can be used to improve the retroviral gene expression in such cells. Such hybrids are obtained by point mutations in the LTRs especially in the U3 region. For example the binding of the ECF-1 repressor can be reduced by a point mutation at -345 of MoMuLV. A point mutation which creates a binding site for SP-1 (e.g. point mutation at -166 (S. McKnight and R. Tijan (1986) (16)) is equally advantageous. It is furthermore advantageous to also introduce mutations outside the LTR regions. It has been shown that an 18 bp region which adjoins the 5'-LTR directly downstream is a negative regulation element (NRE; silencer binding element). Point mutations in this element enable the retroviral transcription to be improved (murine embryonic stem cell virus, MESV; M. Grez et al (1990) (22)).

The object of the invention is to provide retroviral vector hybrids which can be used to further improve retroviral gene expression especially in haematopoietic stem cells and their myeloid (non-lymphatic) derivatives.

The invention concerns retroviral vector hybrids which are characterized in that they

a) contain the U5 region and/or tRNA primer binding site of MESV and/or MoMuSV in the leader region as a U5 region and/or tRNA primer binding site and

b) contain the U3 and R regions from a Friend murine leukemia virus (F-MuLV) as U3 and R regions in 3'-LTR.

The vector hybrids according to the invention preferably contain the leader region of MESV as the leader region. In a further embodiment of the invention the U5 region and/or preferably the tRNA primer binding site can also be derived from MoMuSV.

The vector hybrids according to the invention also preferably contain the LTR from a Friend murine leukemia virus (F-MuLV) as the 3'-LTR and thus also the U5 region from F-MuLV in addition to the U3 and R regions from MoMuLV and MESV.

The vector hybrids can be replication-defective as well as replication-competent.

A replication-defective vector is understood as a vector which contains no retroviral gene functions (gag, env, pol) and therefore cannot independently form virus particles. However, the vector usually contains a packaging function (psi). In order to form infectious retroviral particles a packaging cell line which contains the gene functions gag, env and pol in a stable form (as an episome or integrated into the genome) is transfected with the replication-defective DNA vector according to the invention. RNA transcripts are formed which are packaged into virus particles due to the gag and env functions. These virus particles are replication-deficient because the RNA genome contained in them does not carry retroviral gene functions.

Retroviral vector hybrids capable of replication additionally contain the gene regions gag, pol and env. These gene regions can be derived from any desired retroviruses. Only the selection of the env region depends on the cell to be infected. Such methods are however, known to a person skilled in the art. In this manner it is possible to produce ecotropic, xenotropic, amphotropic or polytropic retroviruses capable of replication.

More details of this and on the production of vectors, virus particles and helper cells are described in M. Kriegler, Gene Transfer and Expression, A Laboratory Manual, W. H. Freeman & Co., New York (1990), 47-55 and 161-164 (31). This publication is a subject matter of the disclosure of the present application.

Silencer proteins within the sense of the invention are understood as cellular (e.g. derived from a mutated cell) nuclear proteins, for example from haematopoietic stem cells, which preferably bind to the first 450 bp preferably the first 18 bp, of MoMuLV and MoMuSV which immediately follow the 5'-LTR and inhibit the retroviral transcription by interaction with cis regulatory sequences of the provirus.

The leader region of MESV is understood as the sum of sequences from the short direct terminal repeat (R), the U5 region and the sequence immediately adjoining the 5'-LTR of MESV which contains the tRNA primer binding site as well as the packaging region (ψ). At a maximum the leader sequence can be composed of the complete sequence which is contained in the virus from R up to the translation start. However, it is preferable to use a truncated leader region which in addition to R and U5 is preferably composed of about the first 450 bases downstream of the end of the 5'-LTR region. It is also preferred that the U5 region and the tRNA primer binding site are derived exclusively or to a major extent from MESV and the other parts of the leader sequence are derived from MoMuLV or MoMuSV. A suitable MESV region is shown in the Figures (FIGS. 1-5) from KpnI (ca. 550) to BalI (ca. 729).

The leader region of MESV corresponds to the leader region of MoMuLV or MoMuSV (sequence of U3, U5 and R of MoMuLV and PCMV, see (22) and (23)) in which point mutations are inserted which reduce or prevent the binding of silencer proteins. Compared to MoMuLV and PCMV the leader region of MESV contains 5 point mutations in the 18 bp region which directly adjoins the 5'-LTR (C. Stocking et al. (1993) (21)). This drastically reduces the inhibitory binding ability of nuclear proteins from the infected cells and correspondingly improves the expression of a gene contained in the vector hybrid (R. Petersen et al., Mol. Cell Biol. 11 (1991) 1213-1221 (8)). Instead of these point mutations, other (at least one) point mutations in the leader region (preferably within the first 450 bases downstream from the end of the 5'-LTR region and particularly preferably in the 18 bp region) are also suitable for reducing or preventing the inhibitory binding of silencer proteins e.g. to the leader region. Thus in addition to the aforementioned sequence described in SEQ ID NO:1 a leader region of MESV within the sense of the invention is also understood as a sequence which essentially corresponds with this section of SEQ ID NO:1, contains a tRNA binding site and a psi function and is modified by point mutations in such a way that the inhibitory binding of silencer proteins from the cells which have been chosen for the transfection is reduced or prevented.

In order to check whether a point mutation is suitable for preventing the binding of silencer proteins to the leader region it is usual to construct a plasmid which contains an LTR, the leader region to be examined and an indicator gene which is preferably the CAT gene. The cells to be examined are transfected with this plasmid and the CAT activity is determined preferably after 48 hours (transient transfection). If the CAT activity is clearly present then the point mutation is suitable for preventing the binding of the silencer protein in these cells (P. Artelt et al. (1991) (32)).

Transient transfection experiments have shown that cis-regulatory sequences are located in the leader sequence of MoMuSV that have an inhibitory influence on the gene expression in particular in early multipotent myeloid cells (primary silencing). A substitution of this region by leader sequences of the murine embryonic stem cell virus MESV (22)! completely abolishes the inhibitory effect in myeloid stem cells. The leader sequence of MESV was originally derived from the endogenous mouse retrovirus dl-587rev (23) and was introduced into MESV in order to abolish the silencing of the MoMuSV leader observed in embryonic stem cells (22). Mutations of the retroviral primer binding site (PBS) which is located immediately 3' of the 5'-LTR are decisive for the abolition of the silencing (22).

Retroviruses of the F-MuLV group are known to a person skilled in the art and are described for example in D. Linnemeyer et al. (1981) (7), J. Friel et al. (1990) (9), S. P. Clark and T. W. Mak (1982) (10), L. Wolff et al. (1985) (11), R. K. Bestwick et al. (1984) (12), W. Koch et al. (1984) (13) and A. Adachi et al. (1984) (14). A review is given in W. Ostertag et al. (1987) (15). The 3'-LTRs of malignant histiosarcoma virus (MHSV) (9), SFFVp Lilly-Steeves (10), SFFVa (11), Rauscher SFFV (12), F-MuLV c157 (13) and Friend-mink cell focus forming virus (F-MCFV FrNx (14), F-MCFV pFM548 (13)) are preferably used.

According to the invention sequences of the genomic vector RNA are suitable as 3'-LTR sequences which have a high degree of sequence homology to the 3'-LTR regions of the aforementioned Friend virus family in the U3 region. The LTR sequence of SFFVp is described in SEQ ID NO:1 (nucleotides 1707-2283).

The advantages according to the invention are exhibited in transient transfections of reporter gene constructs in numerous representative cell lines of humans and mice. It has surprisingly turned out that U3 sequences of mouse retroviruses of the Friend murine leukemia virus (F-MuLV) family in combination with leader sequences (preferably tRNA binding site) of MESV enable an especially efficient gene expression in myeloid stem and precursor cells. The increase of the gene expression with the vector hybrids according to the invention is more than a power of ten compared to MoMUSV. This applies to relatively late precursor cells of the granulocytes, macrophages and erythrocytes as well as to the early multipotent precursors and even stem cells of the myeloid system. The vector hybrids according to the invention enable an efficient gene expression especially in the case of immature multipotent myeloid cells in which MoMuSV-LTR exhibits a pronounced primary silencing. The activity is also high in the lymphatic system as well as in fibroblasts.

An early isolate of the polycythaemia-inducing spleen focus forming virus (SFFVp (7)), is particularly preferred whose LTR sequence is described in SEQ ID NO:1 (bp 2654-3230) as well as the malignant histiosarcoma virus (MHSV (9), LTR SEQ ID NO:4, bp 1707-2324). In the U3 region of these viruses there is a high degree of sequence homology to other representatives of the Friend virus family such as SFFVp Lilly-Steeves (10), SFFVa (11), Rauscher SFFV (12), F-MuLV c 57 (13) and Friend-mink cell focus forming virus F-MCFV FrNx (14), F-MCFV pFM548 (13)!. These and similar Friend-related retroviruses have a similar expression pattern as SFFVp and MHSV due to the sequence homology of the U3 regions.

Mutations in the enhancer region of U3 340 to 140 nucleotides on the 5' side of the transcription start (10)! are particularly advantageous for the pronounced increase compared to MoMuSV of the gene expression in myeloid stem and precursor cells by the vectors according to the invention. The partial duplication of the so-called direct repeat element in SFFVp (similar to F-MCFV FrNx) and MHSV is of importance. Binding sites for transcription factors may be located here which lead to an efficient gene expression in myeloid cells.

SFFVp and MHSV as well as Friend MCFV carry a binding site for the transcription factor Sp1 (16) directly 5' of the direct repeats. Sp1 is capable of multiple interactions with surrounding transcription factors which usually lead to a considerable increase in gene expression. In the case of the MoMuSV derivatives myeloproliferative sarcoma virus (MPSV (17), (18)) and PCC4-cell passaged MPSV (PCMV (19)) the occurrence of an Sp1 binding site in the U3 region is also necessary for the abolition of the silencing of the MoMuSV-LTR in embryonic stem cells (20) as well as probably also in blood stem cells (21). The Sp1 binding site of SFFVp, MHSV as well as Friend-MCF viruses may also have an analogous function.

In a further embodiment of the invention the retroviral vector hybrids can contain the U3 and R regions from MPSV as U3 and R regions in 3'-LTR. MPSV differs from MoMuSV and MoMuLV by point mutations in the LTR (21). In this case A(-381) is deleted and the following substitutions have been carried out C→T, -345; T→A, -326; T→A; -249 and A→C, -166 (numbering according to (21)). In this case the retroviral vector hybrid contains the U5 region and tRNA primer binding site of MESV in the leader region as the U5 region and tRNA primer binding site. It has turned out that such MPSV/MESV hybrid vectors (also called MPEV in the following) have considerable advantages compared to vectors based on MoMuLV and are of major importance for numerous applications in somatic gene therapy.

Any desired U3 and R regions can be used in 5'-LTR since after integration of the virus the U3 from the 3'-LTR is copied in the target cell also at the 5'-LTR position after completion of the retroviral life cycle and drives the gene expression. U3 and R regions are derived for example from MoMuLV or MoMuSV derivatives such as for example MPSV, PCMV and MESV. 5'-LTRs from F-MuLV are also suitable as the 5'-LTR.

The vector hybrids according to the invention exhibit a high tissue-specific expression after retroviral transduction of myeloid stem and precursor cells. These vectors therefore have the potential to considerably increase the gene transfer efficiency in myeloid stem and precursor cells compared to MoMuSV vectors. The vectors according to the invention additionally have the potential to be considerably less subject to silencing processes in myeloid cells than MoMuSV vectors. The high and possibly even persistent gene expression of these vectors in myeloid cells can form the basis for the successful application of numerous gene transfer protocols on the blood-forming system of humans. The constructs described in the examples are constructed in such a way that the cDNAs transferred by the vector can be replaced without difficulty by other genes.

The tissue specificity of the expression is suitable for abolishing the primary silencing in the myeloid system observed in the case of conventional MoMuSV vectors. This leads to a considerable increase in the functional gene transfer rates in myeloid cells. Since a high functional gene transfer rate is desired in most gene transfer/gene therapy applications on the myeloid system this is a generally usable advantage of the constructs according to the invention.

The Friend virus-related U3 regions were also selected in the myeloid system of the mouse for persistency of the gene expression in vivo. Secondary silencing occurred with these vectors to a much lower extent than with MoMuSV vectors. The exclusion of inhibitory leader sequences by substitution for MESV sequences is of additional advantage in this regard. This is of importance for all gene transfer/gene therapy applications in which a persistent (if possible life long) expression of the retrovirally transferred sequences is desired in the myeloid system (gene marker studies, correction of metabolic defects).

For this purpose the vector hybrids according to the invention can contain at least one gene that is heterologous for the virus and can be expressed in eukaryotic cells. Such genes are for example the multiple drug resistance gene (MDR gene), an antibiotic resistance gene such as the neo^(R) gene, the LNGFR gene, the cerebrosidase gene or the herpes simplex TK gene. In a preferred embodiment several and preferably two or three heterologous genes can also be contained in the vector hybrid. In order to enable an expression of these separate genes, it is expedient to insert a promoter, a splicing site (preferably from SF7Vp) or an internal ribosomal entry site (IRES preferably from polio viruses (I. R. Ghattas et al. (1991) (26)) in front of the second or third gene.

The production of a high virus titre in fibroblastoid packaging cell lines is a further important requirement for retroviral vectors which are intended to be used for gene transfer in myeloid cells. In the case of all the construct examples listed below the activity of the Friend-related U3 regions in fibroblastic retrovirus packaging cell lines is adequate to produce the required titre of 10⁵ to 10⁶ vector-transferring retroviral particles/ml cell culture supernatant.

Accordingly a subject matter of the invention is also a process for the production of a retrovirally transduced eukaryotic cell which contains an active exogenous gene which is characterized in that the eukaryotic cell is transduced with a replication-defective retroviral vector virus which in its genome contains

a) the leader region of MESV or MoMuSV as the leader region

b) the U3 and R regions from a Friend murine leukemia virus (F-MuLV) as the U3 and R regions in the 3'-LTR and

c) the said exogenous gene.

The LTR from F-MuLV is preferably used as the 3'-LTR which then contains the said U3 and R regions.

A further subject matter of the invention are retrovirally transduced, eukaryotic cells obtainable by transducing the eukaryotic cell with a replication defective retroviral vector virus which in its genome contains

a) the leader region of MESV and/or MoMuSV as the leader region

b) the U3 and R regions from a Friend murine leukemia virus (F-MuLV) as the U3 and R regions in the 3'-LTR.

This vector optionally contains one or several (up to three) exogenous genes which can be expressed in the eukaryotic cell. Mammalian cells preferably haematopoietic cells and especially haematopoietic stem cells are preferably used as eukaryotic cells. The LTR from F-MuLV is preferably used as the 3'-LTR which then contains the said U3 and R regions.

An active exogenous gene is understood as a gene which is introduced from outside into the cell and is expressed (is active) in this cell after integration into the genome. The exogenous gene may be a gene not present in the cell genome (e.g. an antibiotic resistance gene such as neo^(R), LNGFR receptor (D. Johnson et al. (1986) (33)), the cerebrosidase gene or a herpes simplex TK gene) or a gene present in the genome but not expressed there or only to an inadequate extent such as the multiple drug resistance (MDR) gene.

A further subject matter of the invention is a replication-defective infectious virus particle which contains a retroviral RNA as the genome wherein the genome contains a leader region from MESV which contains a packaging function and a tRNA binding site, and contains a heterologous gene for the virus which can be expressed in a eukaryotic cell and contains at the 3' end U3 and R from a Friend murine leukemia virus (F-MuLV) but no active gag, env and pol sequences.

A further subject matter of the invention is a process for the production of a replication-defective infectious virus particle by transfection of a eukaryotic helper cell which possesses the helper functions gag, env and pol with a vector hybrid which contains the leader region of MESV as the leader region and the LTR from a Friend murine leukemia virus as the 3'-LTR, production of the RNA corresponding to the DNA of the vector hybrid as a virus genome in the cell (for example by cellular polymerases), packaging of the said RNA into the replication-deficient empty virus envelopes that are formed in the cell and isolation of the infectious virus particles which contain the said virus genome.

A further subject matter of the invention is the use of a replication-defective retroviral vector hybrid according to the invention which contains the leader region of MESV as the leader region and the U3 and R regions from a Friend murine leukemia virus (F-MuLV) as the U3 and R regions in the 3'-LTR for the production of a pharmaceutical agent for ex vivo or in vivo gene therapy.

Vector hybrids are also suitable for the process and uses according to the invention which contain the U5 region and/or tRNA binding site of MESV or MoMuLV/MoMuSV in the leader region as the U5 region and/or tRNA binding site.

Areas of Application

I. All somatic gene transfer/gene therapy methods in which myeloid stem cells and precursor cells are the target population for retroviral vectors.

In the individual protocols different cDNAs are integrated into the vectors. Examples are

Gene marker studies (1): Selectable marker genes such as neoR or shortened nerve growth factor receptor are transferred in order to monitor the fate of the labelled cell population in the organism under the conditions of the examined disease/therapy. The neoR vectors pSF1N and pMH1N which carry the neoR under the control of the SFFVp U3 and MHSV U3 (MESV leader) exhibit in comparison to neoR-transferring Maloney vectors a considerable increase in the functional gene transfer rate in the model system of the mouse stem cell line FDCPmix. They are suitable for neoR transfer in human myeloid stem cells.

Protection of bone marrow against the side-effects of a high dose chemotherapy (28): genes mediating chemotherapy resistance such as MDR1 or alkyl transferases are transferred. High transfer rates and expression via the optimized Friend virus-related vectors prevent myelosuppressive side-effects and eventually also secondary tumour induction of the chemotherapy.

Correction of metabolic diseases: In the case of monogenic hereditary diseases intact copies of the defective genes are introduced by means of retroviral gene transfer into myeloid stem cells. Applications are for example conceivable in the case of hereditary diseases whose gene defect has effects on the myeloid system (storage diseases such as the Hurler syndrome; M. Gaucher (29)):

II Cloning of replication-competent Friend-related retroviruses

Due to their extended host spectrum towards early myeloid stem and precursor cells the viruses according to the invention are particularly well suited for insertion mutagenesis in these cells. Insertion mutagenesis enables genes to be cloned which have an important function in the physiological growth regulation of the myeloid system.

The principle of using retroviruses as an insertion mutagen has already been described several times (Review: Kung et al. (1991) (34)). Retroviruses integrate DNA sequences unspecifically into the genome and in this process can activate or inactivate genes. Replicatable retroviruses infect tissues with a very high efficiency and can therefore increase the probability of producing mutations by integration. Such mutations can in turn lead to a degeneration of the mutated cells i.e. tumours are formed.

In in vivo experiments with mice (usually on new-born mice since they do not yet have an immune system which works efficiently and are therefore very sensitive towards retroviruses) for the identification of regulatory genes, these are infected with retroviruses and, after a latency period of about 3 months, it is possible to discover and examine specific tumours (dependent on the virus type). Analyses of the clonal retroviral integration sites reveal genes whose activation or inactivation can lead to cells of a modified or degenerate phenotype. This enables a large number of diverse gene types to be discovered such as genes for ligands, receptors, signal transducers and transcription factors. It is significant that there is a high correlation between the tumour type found which is produced by particular viruses and the activated/inactivated gene (e.g. in the case of erythroleukemia which is induced by Friend-MuLV ca. 80% of the tumours exhibit integration events in the Fli-1 locus and transcriptional activation of the transcription factor PU-1 gene: It was possible to identify interacting oncogenes by infecting transgenic mice with ecotropic expression of an oncogene (e.g. myc) and observing the tumorigenesis. After a considerably shortened latency period tumours were formed by secondary mutations ("second hit" e.g. in the pim-1 gene)). Nevertheless it has only been possible to identify some of the possible genes using the previous retrovirus constructs. The spectrum of genes that can be identified with this approach of activation/inactivation and the concomitant phenotypic modification of the cell depends strongly on the specificity of the retroviruses used for particular cell types. Thus retroviruses that have been previously used only exhibit a very low infectiousness in haematopoietic and embryonic stem cells (cf. Review: Stocking et al. (1993) (21)). This is due to several reasons; primarily to transcription control elements in the retroviral LTRs (Stocking et al. (1985) (18)) and secondarily also due to the protein interactions between cellular and viral proteins that are necessary for viral replication.

Replication-competent retroviruses produce new cells producing retroviruses by infection which leads to a complete infection of the entire cell population and to a much higher probability of forming mutations. A similar efficiency can only be achieved by a much more complicated and time-consuming co-cultivation of the retrovirus packaging line with the target cell line.

Friend-related replication-competent viruses can in addition be used to produce forced passage mutants which also have an additional improvement of the infectiousness towards blood stem cells. Mutations in retroviral structural proteins of these viruses may be used to produce optimized packaging cell lines for gene transfer into myeloid cells. Mutations in cis regulatory sequences of these viruses may be used to further optimize retroviral vectors for blood stem cell gene transfer.

A further subject matter of the invention are ecotropic, xenotropic, amphotropic or polytropic retroviruses capable of replication containing the regulatory elements (leader region) of MESV and optionally MoMuLV or MoMuSV combined with the U3 and R regions from a Friend murine leukemia virus (F-MuLV) as U3 and R regions in the 3'-LTR, which as a result are capable of more efficiently infecting murine (in vivo and in vitro) or human (in vitro) precursor cells and to trigger mutations. The retroviruses preferably contain the LTR from F-MuLV as the 3'-LTR.

The combination of both components leads to retroviral insertion mutagenesis constructs of a new quality which can be used to identify genes which cannot be detected with conventional replicatable retroviruses. They can be produced in a very high titre and result in an almost 100% infection rate since each infected cell becomes a production cell for new viruses. The neomycin resistance gene which is used in retroviral vectors for selection can be deleted in them. This leads to a higher transcription of the LTR promoters since neo^(R) functions as a silencer (Artelt et al. (1991) (32)) and consequently leads to an improved insertion rate.

Murine as well as human haematopoietic and embryonic stem cells can be infected in vitro with a high efficiency by the retroviruses according to the invention and mutants can be produced by insertion into the genome (activation or inactivation of genes). Therefore also in the case of in vivo experiments it would be expected that the viruses according to the invention would infect early haematopoietic and embryonic cells with a higher efficiency and to activate or inactivate genes by integration into the genome.

Gene activation can occur in several ways. Thus integration upstream of a gene can activate it under the transcriptional control of the 3'-LTR promoter. The binding sites of negative control elements can also be inactivated which can lead to a gene activation. There are also several examples (Kung et al. (1991) (34)) which demonstrate that the 5'-LTR promoter can activate a gene after insertion is completed either directly by reading through or by its enhancer effect many bases upstream of and also within an intron.

An integration within a gene or in the accompanying promoter/enhancer region can also lead to an inactivation of the corresponding gene.

If genes are effected which are important for the regulation of the cell it is possible that the cells degenerate i.e. tumours may be formed. The viruses according to the invention can therefore be used to find other genes e.g. genes which are involved in the cell regulation of haematopoietic or embryonic stem cells which are different from those that can be found by conventional viruses which do not replicate or only very poorly in such cell types.

The replication-competent viruses according to the invention can also be used in vitro for example in the following manner:

Factor-dependent promyelocytic cell lines were made factor-independent by retroviral insertion mutagenesis i.e. the growth factor gene was activated by retroviral integration. The retrovirus infection was achieved by co-culturing the target cell line with packaging lines producing the MPSV vector. This in vitro approach can be made more efficient by using the constructs according to the invention. Moreover murine or human embryonic and haematopoietic stem cells can also be used in the above investigations using the viruses according to the invention.

A further application is in experiments which have the goal of producing improved vector virus packaging lines, e.g. for gene therapy, which can produce viruses with a higher infection efficiency in precursor cells. In these cells there are several blocks which interfere with the retroviral infection/replication. The use of the constructs according to the invention has the advantage over replicatable wild-type MoMuLV or AM4070 that the transcriptional blocks are already removed. Improvements in the infection efficiency can therefore be attributed to corresponding mutations in the viral proteins.

A further application of the constructs according to the invention is to produce "knock out" mice by insertion inactivation of one or several genes in embryonic stem cells.

The invention is further elucidated by the following examples, diagrams and sequence protocols.

The sequence protocols show:

SEQ ID NO:1 sequence of pSF1

SEQ ID NO:2 sequence of pSF2

SEQ ID NO:3 sequence of pSF3

SEQ ID NO:4 sequence of pMH1

SEQ ID NO:5 sequence of pSF-MDR

SEQ ID NO:6 sequence of pMP-MDR

(only the proviral part of the sequences is shown in each case)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the plasmid map of pSF-MDR: (information on restriction sites for guidance)

FIG. 1B shows the plasmid map of pSF-MDR (more exact details of restriction sites)

FIG. 2A shows the restriction map of pSF1

FIG. 2B shows the restriction map of pSF1N. This vector corresponds to pSF1 into which the neo^(R) gene was inserted into the multiple cloning site (MCS).

FIG. 3A shows the restriction map of pSF2

FIG. 3B shows the restriction map of pSF2N which corresponds to pSF2 after integration of the neo^(R) gene

FIG. 4A shows the restriction map of pSF3

FIG. 4B shows the restriction map of pSF3N which corresponds to pSF3 after integration of the neo^(R) gene

FIG. 5A shows the restriction map of pMH1

FIG. 5B shows the restriction map of pMH1N which corresponds to pMh1 after integration of the neo^(R) gene.

FIG. 6 shows the scheme for the formation of vector pSF-MDR1.

FIG. 7 shows the scheme for the formation of vectors pSF1N, pSF2N, pSF3N and pMh1N.

FIG. 8 shows the protection of myeloid cells by FMEV and MPEV-mdr1 vectors in comparison to standard MoMuLV vectors. The average relative transduction frequency is stated (corrected for fibroblast titres and cloning efficiency). Fibroblast titres between the various vectors vary by less than factor 3. A: cell line K-562, B: cell line TF-1.

FIG. 9 shows the restriction map of pMP-MDR.

EXAMPLE 1 a) Description of construction

Table 1 shows the cloning strategies of the constructs according to the invention.

I. pSF-MDR (FIGS. 1, 6, Table 1, SEQ ID NO:5)

This vector was cloned based on the MESV vector R224 (base pUC 19) in which parts of the env region as well as the complete U3 region of the 3'LTR were replaced by SFFV-p sequences. Vector pSF5+3 (cf. FIG. 6) was obtained in this manner. In a second step the cDNA (from pMDR2000XS (25)) coding for MDR1 was inserted into the multiple cloning site (NotI, XbaI, BamHI, Hind3).

II. pSF1N, pSF2N, pSF3N, pMH1N (FIGS. 2-5, 7, Table 1)

The backbone of these plasmids is based on the MESV vector R224 in which a major deletion had been carried out between the Xba1 cleavage site of the the 5'LTR and the Kpn1 cleavage site in the 3'LTR. The complementing sequences were inserted by means of three fragment ligations; base vectors pSF1, pSF2, pSF3 and pMH1 result (formation scheme and cloning strategy, FIG. 7, Table 1, sequences described in SEQ ID NO:1-4). The fragments used for the three fragment ligation are obtained from auxiliary constructs (see formation scheme, FIG. 6) in which simple modifications can be carried out in order to further optimize the vectors. It is also conceivable to substitute the SFFVp or MHSV U3 regions by analogous sequences of other Friend-related retroviruses or by the above-mentioned modifications of the leader in order to optimize the titre. The construct examples pSF 1N, pSF2N, pSF3N, pMh1N were obtained after insertion of the neoR cDNA (from R229). The construct examples carry polylinkers with singular restriction cleavage sites which can be used for the exchange of genes to be transferred. At these cleavage sites it is additionally possible to insert regulatory sequences for secondary gene expression that may be necessary such as the splice acceptor of SFFVp or the so-called internal ribosome entry site IRES (I. R. Ghattas et al. (1991) (26)).

The Friend-related regions cloned in the construct examples as U3 and R (in the 3'-LTR) are also copied at the 5'-LTR position after completion of a retroviral life cycle and then drive gene expression in the target cells. The construct examples contain all cis regulatory elements necessary for retroviral gene transfer and retroviral gene expression. Properties of SFFVp (or MHSV), MESV and MoMuSV are combined in these constructs. The point mutation of the start codon for the retroviral gag protein to form a stop codon (A. D. Miller and G. J. Rosman (1990) (27)) and the deletion of superfluous env sequences (27) are integrated as safety-relevant modifications in pSF1N, pSF2N, pSF3N, pMH1N.

                                      TABLE 1     __________________________________________________________________________     Cloning strategies for the constructs and     auxiliary constructs according to the     invention.     Construct           Backbone    Fragment 1  Fragment 2     __________________________________________________________________________     pSFMDR           SF5 + 3 Sal1/not1                       pMDRSX1 Sal1/Not1     pSF5 + 3           R224ΔNB Xho 1/Nru1                       psFpolyHind3 blunt end     pMDRΔSX1           bluescript KS Sal1/Sma1                       pMDR2000XS Sal1/Ssp1     pSF1N pSF1 Not1/BamH1                       neoR Not1/BamHI (R229)     pSF2N pSF2 Not1/BamH1                       neoR Not1/BamHI (R229)     pSF3N pSF3 Not1/BamH1                       neoR Not1/BamHI (R229)     pMH1N pMH1 Not1/BamH1                       neoR Not1/BamHI (R229)     pSF1  R229ΔXba1/Kpn1                       pLl Xba1/Not1                                   pΔenvSF Not1/Kpn1     pSF2  R229ΔXba1/Kpn1                       pL2 Xba1/Not1                                   pΔenvSF Not1/Kpn1     pSF3  R229ΔXba1/Kpn1                       pL3 Xba1/Not1                                   pΔenvSF Not1/Kpn1     pMH1  R229ΔXba1/Kpn1                       pLl Xba1/Not1                                   pΔenvMH Not1/Kpn1     pL1   bluescript KS Xba1/Not1                       R224 Xba1/Not 1     pL2   pL1ΔBal1                       pV-MDR 1 Bal1     pL3   pL1ΔKpn1-Pst1                       pV-MDR1 Kpn1/Pst1     pΔenvMO           bluescript KSBamH1/Kpn1                       pV-MDR1 BamH1/Kpn1     pΔenvSF           pΔenvMo Nhe1/Kpn1                       pSFU3 Xba1/Kpn1     pΔenvMH           pΔenvSF EcoR5/Kpn1                       pBR-MHSV EcoR5/Kpn1     pSFU3 pUC19 Hinc2 pSFenv SfaN1/Kpn1     __________________________________________________________________________

EXAMPLE 2 Increasing the functional gene transfer rate in comparison to conventional Moloney vectors

The multiple drug resistance 1 (MDR1) gene codes for an efflux pump located in the membrane (P-glycoprotein) which mediates resistance to a series of clinically important cytostatic agents (I. Pastan et al. (1988) (25)). The degree of resistance is closely related to the level of expression of P-glycoprotein. If myeloic stem cells are successfully made resistant to cytostatic agents by retroviral MDR1 transfer, this could make an important contribution to lowering the side-effect rate in tumour chemotherapy.

In the experiment K562 cells (ATCC CLL243) and TF-1 cells (human myeloid progenitor cell lines) were infected with retroviral vectors which express MDR1 under the control of the SFFV-LTR (virus SF-MDR resulting from the construct pSF-MDR with MESV leader and MPSV/MESV hybrid vector pMP-MDR).

The TF1 cell line was obtained from a patient with erythroleukemia as described by T. Kitamura et al. (1989) (30). The cells are kept in culture in RPMI medium (supplemented with 20% foetal calf serum, 1 mM Na pyruvate, 4 mM glutamine and 20 U/ml recombinant GM-CSF).

K562 cells and TF1 cells were infected in an identical mixture with a Moloney-MDR1 vector (virus V-MDR resulting from the construct pVMDR) which is used in a clinical gene transfer protocol under the leadership of Prof. A. Deisseroth at the MD Anderson Cancer Center, Houston, Tex. As can be seen from Table 2 SF-MDR in the case of K562 leads to a 20-fold increase of the gene transfer rate compared to VMDR as measured by the number of colonies growing when cytostatic agents are administered (ca. 10-fold LD50 for K562).

The increase of the functional gene transfer rate in myeloid cells is an expression of the average increase of the gene expression rate in transduced cells. This is important in gene transfer applications whose success also depends on the level of gene expression (see example of application: protection of bone marrow in high-dose chemotherapy). If the cells are plated out with very high doses of cytostatic agents (ca. 20-fold LD50 for TF1) in the experiment described above (infection of human myeloid cells with SFMDR or V-MDR) only cells infected with SF-MDR are able to form colonies (Table 2).

                  TABLE 2     ______________________________________     Colony number under colchicine selection after     infection of human haematopoietic cells K562 and TF1 by     MDR1-transduced retroviral vectors. The vectors pSF, MDR     and pMP-MDR considerably increase the functional gene     transfer rate compared to a conventional MoMuLV vector.                       Number of colonies under                       selection             U3 region           K562 (20 ng                                          TF1 (80 ng     Virus   of 3'LTR  Leader    colchicine)                                          cochicine)     ______________________________________     SF-MDR  SFFVp     MESV      948 +/- 98                                          13 +/- 3     V-MDR   MoMuLV    MoMuSV    38 +/- 4 0     MP-MDR  MPSV      MESV      410 ± 28                                          9 ± 3     ______________________________________

The cells were infected with a retroviral supernatant of amphotropic packaging cell lines. The titre of fibroblasts was ca. 2×10⁴ /ml for both vectors at the time of the experiment. The ratio of vector/target cell was <1 during infection. 24 hours after the infection 5×10⁴ cells were plated out in soft agar medium containing colchicine. The evaluation was carried out on day 8 of the agar cloning. The cloning efficiency without selection was ca. 15% for both cell lines. The experiments were carried out in duplicate.

Analogous results are obtained to those of vector pV-MDR for a vector whose U3 region is derived from MoMuLV and whose leader is derived from MESV.

In the MDR1 system constructs with an MESV leader have slightly reduced titres compared to conventional MoMuSV vectors. This may be due to mutations in the packaging region of MESV. Hybrids between the leader sequences of MESV and MoMuSV have the potential to produce vectors with a higher titre due to an improved packaging function in retroviral packaging cell lines with simultaneous exclusion of cis-inhibitory sequences. This was taken into account when cloning the construct examples pSF2N and pSF3N.

Note: Proviral vectors are for example denoted SF-MDR, MP-MDR etc. in order to differentiate them from the corresponding plasmids pSF-MDR, pMP-MDR etc.

EXAMPLE 3 Construction of insertion mutagenesis vectors capable of replication based on the Friend/MESV vector constructs according to the invention

The construct example of an amphotropic Friend/MESV vector capable of replication was produced for insertion mutagenesis by cloning the gag-pol-env genes necessary for the retroviral replication into the Friend-/MESV vector pSF1 and pSF3 (see FIGS. 2A and 4A).

For this the entire 7.2 kb gag-pol-env gene cassette from pAM (Miller et al. (1985) (35)) was amplified with the help of the "expand long template PCR system" (Boehringer Mannheim, see also: W. M. Barns (1994) (36)) and incorporated into pSF1 and pSF3.

pAM represents a fusion construct from the ecotropic MoMuLV vector pMLV-K (gag-pol to the SalI cleavage site) and from the amphotropic virus 4070A vector p4070A (pol from the SalI cleavage site and env^(amphotropic)). The upper PCR primer was placed 60 bp upstream of the gag start codon (directly adjoining the PstI cleavage site), the lower PCR primer was directly placed at the end of the envamphotropic gene. The 7.2 kb gag-pol-env^(amphotropic) PCR amplificate was incorporated uncleaved i.e. with blunt ends, into the vectors pSF1 and pSF3 that had been cleaved with PstI and HindIII and treated with Klenow enzyme in order to remove overhangs (Sambrook et al. (1989) (37)). The resulting vectors were named pSF1-AM, pSF3-AM and are in principle constructed as follows: 5 1'-LTR^(MESV) -PBS(-)^(MESV) -gag-pol-env-^(amphotropic) -3'-LTR^(SFFV) (pSF1-AM) and 5'-LTR^(MESV) -PBS(-)^(MUSV) -gag-pol-env^(amphotropic) -3'-LTR-^(SFFV) (pSF3-AM).

After transfection in human or murine cells they are able to form replication-competent retroviruses (RCR) which can be used for insertion mutagenesis in haematopoietic stem and progenitor cells.

EXAMPLE 4 Construction of insertion mutagenesis vectors capable of replication based on MPSV/MESV vector constructs according to the invention

In the vector MoMuLV-TAT (Hilberg, F. et al. Prog. Natl. Acad. Sci. USA 84 (1987) 5232-5236 (38) the NheI-KpnI fragment was replaced by the corresponding LTR fragment of MPSV (-416-+31). The resulting vector was named MP-CAT. This plasmid can be used as a reporter gene plasmid by expressing the chloroamphenicol transferase (CAT) gene under the control of the retroviral LTR fragment.

The vector P50-M is used as the MPSV/MESV base vector. This vector contains the MPSV-U3 in the 3'-LTR. It is a derivative of p5Gneo which contains a 537 bp long leader fragment of dl587rev (Grez M. et al., Proc. Natl. Acad. Sci. USA 87 (1990), 9202-9206 (39), Colicelli J. and Goff S. P., J. Virol. 57 (1987) 37-45 (40)). NeoR-env coding regions of p5Gneo were replaced by a polylinker from p Bluescript KS (Stratagene) and the AUG of gag was destroyed by point mutation. The mdr-1 cDNA was cut out from pMDR2000XS (base pair -138-+3878) as a SacI/SspI fragment (Chen C. et al., Cell 47 (1986) 381-389 (41)). After subcloning in p Bluescript II KS the cDNA was isolated as a BamHI fragment and p50-M was inserted. The vector produced in this way is named pMP-MDR. The sequence of p-MDR, pMP-MDR is shown in FIG. 9 and SEQ ID NO:6.

List of references

(1) Miller A. D., Nature 357, 455-460 (1992)

(2) Mulligan R. C., Science 260, 926-932 (1993)

(3) Vile R. and Russell S. J., Gene Therapy 1, 88-98 (1994)

(4) Lu M. et al., Human Gene Therapy 5, 203-208 (1994)

(5) Palmer T. D. et al., Proc. Natl. Acad. Sci. U.S.A. 88, 1330-1334 (1991)

(6) Brenner M. K. et al., The Lancet 342, 1134-1137 (1993)

(7) Linemeyer D. L. et al., Proc. Natl. Acad. Sci. U.S.A. 78, 1401-1405 (1981)

(8) R. Petersen et al., Mol. Cell. Biol. 11, 1213-1221 (1991)

(9) Friel J. et al., J. Virol. 64, 369-378 (1990)

(10) Clark S. P. and Mak T. W., Nucl. Acid Res. 10, 3315-3330 (1982)

(11) Wolff L. et al., J. Virol. 53, 570-578 (1985)

(12) Bestwick R. K. et al., J. Virol. 51, 695-705 (1984)

(13) Koch W. et al., J. Virol. 49, 828-840 (1984)

(14) Adachi A. et al., J. Virol. 50, 813-821 (1984)

(15) Ostertag W. et al., Adv. Cancer Res. 48, 193-355 (1987)

(16) McKnight S. and Tijan R., Cell 46, 795-805 (1986)

(17) Ostertag W. et al., J. Virol. 33, 573-582 (1980)

(18) Stocking C. et al., Proc. Natl. Acad. Sci. U.S.A. 82, 5746-5750 (1985)

(19) Franz T. et al., Proc. Natl. Acad. Sci. U.S.A. 83, 3292-3296 (1986)

(20) Grez M. et al., J. Virol. 65, 4691-4698 (1991)

(21) Stocking C. et al., in Virus Strategies, ed. by W. Doerfler and P. Bohm, VCH Verlagsgesellschaft, Weinheim, Germany (1993)

(22) Grez M. et al., Proc. Natl. Acad. Sci. U.S.A. 87, 9202-9206 (1990)

(23) Colicelli J. and Goff S. P., J. Virol. 57, 37-45 (1987)

(24) Bilello J. A. et al., Virology 107, 331-344 (1980)

(25) Pastan I. et al., Proc. Nat. Acad. Sci. USA 85, 4486-4490 (1988)

(26) Ghattas I. R. et al., Mol. Cell. Biol. 11, 5848-5859 (1991)

(27) Miller A. D. and Rosman G. J., Biotechniques 7, 980-990 (1990)

(28) Anderson W. F., Human Gene Therapy 5, 1-2 (1994)

(29) Ohashi T. et al., Proc. Nat. Acad. Sci. USA 89, 11332-11336 (1992)

(30) T. Kitamura et al., J. Cell. Physiol. 140, 323-334 (1989)

(31) M. Kriegler, Gene Transfer and Expression, A Laboratory Manual, W. H. Freeman & Co., New York (1990), 47-55 and 161-164

(32) P. Artelt et al., Gene 99, 249-254 (1991)

(33) D. Johnson et al., Cell 47, 545-554 (1986)

(34) H. J. Kung et al., Current Topics in Microbiol. & Immunol. 171 (1991) 1-25

(35) A. D. Miller et al., Molecular and Cell Biology, Vol. 5, No. 3 (1985) 431-437

(36) W. M. Barns, Proc. Natl. Acad. Sci. USA 91 (1994) 2216-2220

(37) J. Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition (1989), CSH Laboratory Press

(38) Hilberg, F. et al, Prog. Natl. Acad. Sci. USA 84 (1987) 5232 -5236

(39) Grez M. et al., Proc. Natl. Acad. Sci. USA 87 (1990), 9202-9206

(40) Colicelli J. und Goff S. P., J. Virol. 57 (1987) 37-45

(41) Chen C. et al., Cell 47 (1986) 381-389

    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 6     (2) INFORMATION FOR SEQ ID NO: 1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5323 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: circular     (ii) MOLECULE TYPE: DNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:     CGATTAGTCCAATTTGTTAAAGACAGGATATCAGGTGGTCCAGGCTCTAGTTTTGACTCA60     ACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAGAATAAAAGATTTTATT120     TAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTA180     AGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCA240     AGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTT300     CCTGCCCCGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTT360     TCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTGCCT420     TATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAG480     CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGCGTCG540     CCCGGGTACCCGTATTCCCAATAAAGCCTCTTGCTGTTTGCATCCGAATCGTGGACTCGC600     TGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCACCTCGGGGGTCTTTCATTTG660     GAGGTTCCACCGAGATTTGGAGACCCCAGCCCAGGGACCACCGACCCCCCCGCCGGGAGG720     TAAGCTGGCCAGCGGTCGTTTCGTGTCTGTCTCTGTCTTTGTGCGTGTTTGTGCCGGCAT780     CTAATGTTTGCGCCTGCGTCTGTACTAGTTGGCTAACTAGATCTGTATCTGGCGGTCCCG840     CGGAAGAACTGACGAGTTCGTATTCCCGGCCGCAGCCCCTAGGAGACGTCCCAGCGGCCT900     CGGGGGCCCGTTTTGTGGCCCGTTCTGTGTCGTTAACCACCCGAGTCGGACTTTTTGGAG960     CTCCGCCACTGTCCGAGGGGTACGTGGCTTTGTTGGGGGACGAGAGACAGAGACACTTCC1020     CGCCCCCGTCTGAATTTTTGCTTTCGGTTTTACGCCGAAACCGCGCCGCGCGTCTTGTCT1080     GCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATT1140     AGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCTTAGATCACTGGAAAGATGTCGAGC1200     GGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGATGGGTTACCTTCTGCTCTG1260     CAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCA1320     TCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTCC1380     CCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTG1440     TACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTC1500     CTCTTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGGCT1560     CCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCAAGCTTATCGATAGGCCTAGGCC1620     TATCGATAGGCCTAGGCCTATCGATAGGCCTAACACGAGCCATAGATAGAATAAAAGATT1680     TTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTA1740     GAGTCGCTTAGCCTGATAGCCGCAGTAACGCCATTTTGCAAGGCATGGAAAAATACCAAA1800     CCAAGAATAGGGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAA1860     ACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCAAGAACAGATGGTCACCG1920     CAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCA1980     GCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTG2040     CGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTC2100     CCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTG2160     AGTCGCCCGGGTACCCGTGTTCTCAATAAACCCTCTTGCAGTTGCATCCGACTCGTGGTC2220     TCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTC2280     AGTTTCTCCCACCTACACAGGTCTCACTAACATTCCTGATGTGCCGCAGGGACTCCGTCA2340     GCCCGGTTTGTGTTTATAATAAAATGCAAGAACAGTGTTCCCTTCAAGCCAGACTACATC2400     CTGACTCTCGGCTTTATAAAAGAATGTTGAAGGGCTCTGTGGACTATCTGCCACACGACT2460     TTTAAGATTTTTATGCCTCCTGGATGAGGGATTTAGTCAATCTATCCTCGTCTATTTTGC2520     TGGCTTCTCCGTATTTTAAATTTCTAGTTTGCACTCCCTTCCTGAGAGCACGGCGATTGC2580     AGAGTAGTTAATACTCTGAGGGCAGGCTTCTGTGAAAAGGTTGCCTGGGCTCAGTGTGAG2640     ATTTTGCCATAAAAAGGGGTCCTGCCCCTGTGTACAGACAGATCGGAATCTAGAGTGCAT2700     ACTCAGAGTCCCCGCGGTTCCGGGGCTCTGATCTCAGGGCATCTTTGCCTAGAGATCCTC2760     TACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTAT2820     ATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGT2880     TTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTG2940     CATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTC3000     CTAATGCAGGAGTCGCATAAGGGAGAGCGTCCTGCATTAATGAATCGGCCAACGCGCGGG3060     GAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTC3120     GGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCAC3180     AGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAA3240     CCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCA3300     CAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC3360     GTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATA3420     CCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTA3480     TCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCA3540     GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGA3600     CTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG3660     TGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGG3720     TATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGG3780     CAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG3840     AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAA3900     CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGAT3960     CCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTC4020     TGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTC4080     ATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATC4140     TGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGC4200     AATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTC4260     CATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTT4320     GCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGC4380     TTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA4440     AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTT4500     ATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATG4560     CTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACC4620     GAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAA4680     AGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT4740     GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTT4800     CACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAG4860     GGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTA4920     TCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT4980     AGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTAT5040     CATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGG5100     TGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTA5160     AGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCG5220     GGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTG5280     TGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGG5323     (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5294 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: circular     (ii) MOLECULE TYPE: DNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:     CGATTAGTCCAATTTGTTAAAGACAGGATATCAGGTGGTCCAGGCTCTAGTTTTGACTCA60     ACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAGAATAAAAGATTTTATT120     TAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTA180     AGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCA240     AGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTT300     CCTGCCCCGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTT360     TCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTGCCT420     TATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAG480     CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGCGTCG540     CCCGGGTACCCGTATTCCCAATAAAGCCTCTTGCTGTTTGCATCCGAATCGTGGACTCGC600     TGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCACCTCGGGGGTCTTTCATTTG660     GAGGTTCCACCGAGATTTGGAGACCCCAGCCCAGGGACCACCGACCCCCCCGCCGGGAGG720     TAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGTTTGATGTTA780     TGCGCCTGCGTCTGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAA840     CTGACGAGTTCTGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTTGGGGGCC900     GTTTTTGTGGCCCGACCTGAGGAAGGGAGTCGATGTGGAATCCGACCCCGTCAGGATATG960     TGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTT1020     CGGTTTGGAACCGAAGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGT1080     CTGACTGTGTTTCTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGT1140     TTGACCTTAGATCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTC1200     AAGAAGAGACGATGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGG1260     CCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCA1320     CCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCT1380     TTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCT1440     CCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCTTTCGACCCCGCCTCGATCCTCCCTT1500     TATCCAGCCCTCACTCCTTCTCTAGGCGGCTCCACCGCGGTGGCGGCCGCTCTAGAACTA1560     GTGGATCCAAGCTTATCGATAGGCCTAGGCCTATCGATAGGCCTAGGCCTATCGATAGGC1620     CTAACACGAGCCATAGATAGAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAAT1680     GAAAGACCCCACCTGTAGGTTTGGCAAGCTAGAGTCGCTTAGCCTGATAGCCGCAGTAAC1740     GCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGGGAAGTTCAGATCAAGGGC1800     GGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGC1860     CCCGGCCCGGGGCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAA1920     CAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTC1980     CAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTG2040     CTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCC2100     TCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTTCTCAATAA2160     ACCCTCTTGCAGTTGCATCCGACTCGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGA2220     GTGATTGACTACCCGTCAGCGGGGGTCTTTCAGTTTCTCCCACCTACACAGGTCTCACTA2280     ACATTCCTGATGTGCCGCAGGGACTCCGTCAGCCCGGTTTGTGTTTATAATAAAATGCAA2340     GAACAGTGTTCCCTTCAAGCCAGACTACATCCTGACTCTCGGCTTTATAAAAGAATGTTG2400     AAGGGCTCTGTGGACTATCTGCCACACGACTTTTAAGATTTTTATGCCTCCTGGATGAGG2460     GATTTAGTCAATCTATCCTCGTCTATTTTGCTGGCTTCTCCGTATTTTAAATTTCTAGTT2520     TGCACTCCCTTCCTGAGAGCACGGCGATTGCAGAGTAGTTAATACTCTGAGGGCAGGCTT2580     CTGTGAAAAGGTTGCCTGGGCTCAGTGTGAGATTTTGCCATAAAAAGGGGTCCTGCCCCT2640     GTGTACAGACAGATCGGAATCTAGAGTGCATACTCAGAGTCCCCGCGGTTCCGGGGCTCT2700     GATCTCAGGGCATCTTTGCCTAGAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCAC2760     CGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCG2820     GGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGT2880     GGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCT2940     CAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCG3000     TCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTT3060     CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAG3120     CTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA3180     TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT3240     TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC3300     GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCT3360     CTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCG3420     TGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA3480     AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACT3540     ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA3600     ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTA3660     ACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT3720     TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTT3780     TTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA3840     TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCA3900     TGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAAT3960     CAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG4020     CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT4080     AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAG4140     ACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGC4200     GCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAG4260     CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCA4320     TCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAA4380     GGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA4440     TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATA4500     ATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCA4560     AGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGG4620     ATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGG4680     GGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTG4740     CACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAG4800     GAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC4860     TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA4920     TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAG4980     TGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTA5040     TCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGC5100     AGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTC5160     AGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGC5220     AGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAA5280     AATACCGCATCAGG5294     (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5292 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: circular     (ii) MOLECULE TYPE: DNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:     CGATTAGTCCAATTTGTTAAAGACAGGATATCAGGTGGTCCAGGCTCTAGTTTTGACTCA60     ACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAGAATAAAAGATTTTATT120     TAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTA180     AGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCA240     AGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTT300     CCTGCCCCGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTT360     TCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTGCCT420     TATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAG480     CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGCGTCG540     CCCGGGTACCGTATTCCCAATAAAGCCTCTTGCTGTTTGCATCCGAATCGTGGTCTCGCT600     GTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCACGACGGGGGTCTTTCATTTGG660     GGGCTCGTCCGGGATTTGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTA720     AGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGTTTGATGTTATG780     CGCCTGCGTCTGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACT840     GACGAGTTCTGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTTGGGGGCCGT900     TTTTGTGGCCCGACCTGAGGAAGGGAGTCGATGTGGAATCCGACCCCGTCAGGATATGTG960     GTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCG1020     GTTTGGAACCGAAGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCT1080     GACTGTGTTTCTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTT1140     GACCTTAGATCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAA1200     GAAGAGACGATGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCC1260     GCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACC1320     TGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTT1380     TGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCC1440     ATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCTTTCGACCCCGCCTCGATCCTCCCTTTA1500     TCCAGCCCTCACTCCTTCTCTAGGCGGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGT1560     GGATCCAAGCTTATCGATAGGCCTAGGCCTATCGATAGGCCTAGGCCTATCGATAGGCCT1620     AACACGAGCCATAGATAGAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGA1680     AAGACCCCACCTGTAGGTTTGGCAAGCTAGAGTCGCTTAGCCTGATAGCCGCAGTAACGC1740     CATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGGGAAGTTCAGATCAAGGGCGG1800     GTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCC1860     CGGCCCGGGGCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACA1920     GATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCA1980     GGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCT2040     TCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTC2100     ACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTTCTCAATAAAC2160     CCTCTTGCAGTTGCATCCGACTCGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGT2220     GATTGACTACCCGTCAGCGGGGGTCTTTCAGTTTCTCCCACCTACACAGGTCTCACTAAC2280     ATTCCTGATGTGCCGCAGGGACTCCGTCAGCCCGGTTTGTGTTTATAATAAAATGCAAGA2340     ACAGTGTTCCCTTCAAGCCAGACTACATCCTGACTCTCGGCTTTATAAAAGAATGTTGAA2400     GGGCTCTGTGGACTATCTGCCACACGACTTTTAAGATTTTTATGCCTCCTGGATGAGGGA2460     TTTAGTCAATCTATCCTCGTCTATTTTGCTGGCTTCTCCGTATTTTAAATTTCTAGTTTG2520     CACTCCCTTCCTGAGAGCACGGCGATTGCAGAGTAGTTAATACTCTGAGGGCAGGCTTCT2580     GTGAAAAGGTTGCCTGGGCTCAGTGTGAGATTTTGCCATAAAAAGGGGTCCTGCCCCTGT2640     GTACAGACAGATCGGAATCTAGAGTGCATACTCAGAGTCCCCGCGGTTCCGGGGCTCTGA2700     TCTCAGGGCATCTTTGCCTAGAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCG2760     GCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGG2820     CTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGG2880     CCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCA2940     ACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTC3000     CTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC3060     GCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCT3120     CACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG3180     TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC3240     CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA3300     AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT3360     CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG3420     GCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAG3480     CTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT3540     CGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAAC3600     AGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAAC3660     TACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTC3720     GGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTT3780     TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC3840     TTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATG3900     AGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA3960     ATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCA4020     CCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAG4080     ATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAC4140     CCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGC4200     AGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCT4260     AGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATC4320     GTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGG4380     CGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATC4440     GTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAAT4500     TCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAG4560     TCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGAT4620     AATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG4680     CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCA4740     CCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGA4800     AGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTC4860     TTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATA4920     TTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTG4980     CCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATC5040     ACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAG5100     CTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAG5160     GGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAG5220     ATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAA5280     TACCGCATCAGG5292     (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5364 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: circular     (ii) MOLECULE TYPE: DNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:     CGATTAGTCCAATTTGTTAAAGACAGGATATCAGGTGGTCCAGGCTCTAGTTTTGACTCA60     ACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAGAATAAAAGATTTTATT120     TAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTA180     AGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCA240     AGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTT300     CCTGCCCCGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTT360     TCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTGCCT420     TATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAG480     CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGCGTCG540     CCCGGGTACCCGTATTCCCAATAAAGCCTCTTGCTGTTTGCATCCGAATCGTGGACTCGC600     TGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCACCTCGGGGGTCTTTCATTTG660     GAGGTTCCACCGAGATTTGGAGACCCCAGCCCAGGGACCACCGACCCCCCCGCCGGGAGG720     TAAGCTGGCCAGCGGTCGTTTCGTGTCTGTCTCTGTCTTTGTGCGTGTTTGTGCCGGCAT780     CTAATGTTTGCGCCTGCGTCTGTACTAGTTGGCTAACTAGATCTGTATCTGGCGGTCCCG840     CGGAAGAACTGACGAGTTCGTATTCCCGGCCGCAGCCCCTAGGAGACGTCCCAGCGGCCT900     CGGGGGCCCGTTTTGTGGCCCGTTCTGTGTCGTTAACCACCCGAGTCGGACTTTTTGGAG960     CTCCGCCACTGTCCGAGGGGTACGTGGCTTTGTTGGGGGACGAGAGACAGAGACACTTCC1020     CGCCCCCGTCTGAATTTTTGCTTTCGGTTTTACGCCGAAACCGCGCCGCGCGTCTTGTCT1080     GCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATT1140     AGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCTTAGATCACTGGAAAGATGTCGAGC1200     GGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGATGGGTTACCTTCTGCTCTG1260     CAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCA1320     TCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTCC1380     CCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTG1440     TACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTC1500     CTCTTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGGCT1560     CCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCAAGCTTATCGATAGGCCTAGGCC1620     TATCGATAGGCCTAGGCCTATCGATAGGCCTAACACGAGCCATAGATAGAATAAAAGATT1680     TTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTA1740     GAGTCGCTTAGCCTGATAGCCGCAGTAACGCCATTTTGCAAGGCATGGAAAAATACCAAA1800     CCAAGAATAGGGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAA1860     ACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCAAGAACAGATGGTCACCG1920     CAGTTTCGGCCCCGGCCCGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCG1980     GGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCAT2040     CAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACC2100     AATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGC2160     TCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTG2220     TTCTCAATAAACCCTCTTGCAGTTGCATCCGACTCGTGGTCTCGCTGTTCCTTGGGAGGG2280     TCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCAGTTTCTCCCACCTACACA2340     GGTCTCACTAACATTCCTGATGTGCCGCAGGGACTCCGTCAGCCCGGTTTGTGTTTATAA2400     TAAAATGCAAGAACAGTGTTCCCTTCAAGCCAGACTACATCCTGACTCTCGGCTTTATAA2460     AAGAATGTTGAAGGGCTCTGTGGACTATCTGCCACACGACTTTTAAGATTTTTATGCCTC2520     CTGGATGAGGGATTTAGTCAATCTATCCTCGTCTATTTTGCTGGCTTCTCCGTATTTTAA2580     ATTTCTAGTTTGCACTCCCTTCCTGAGAGCACGGCGATTGCAGAGTAGTTAATACTCTGA2640     GGGCAGGCTTCTGTGAAAAGGTTGCCTGGGCTCAGTGTGAGATTTTGCCATAAAAAGGGG2700     TCCTGCCCCTGTGTACAGACAGATCGGAATCTAGAGTGCATACTCAGAGTCCCCGCGGTT2760     CCGGGGCTCTGATCTCAGGGCATCTTTGCCTAGAGATCCTCTACGCCGGACGCATCGTGG2820     CCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATG2880     GGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGG2940     CAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGG3000     CGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATA3060     AGGGAGAGCGTCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATT3120     GGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA3180     GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCA3240     GGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG3300     CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGT3360     CAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC3420     CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCT3480     TCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC3540     GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA3600     TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA3660     GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG3720     TGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAG3780     CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGT3840     AGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA3900     GATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG3960     ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA4020     AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA4080     ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC4140     CCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATG4200     ATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGA4260     AGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGT4320     TGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT4380     GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC4440     CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTC4500     GGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCA4560     GCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG4620     TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCG4680     TCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAA4740     CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA4800     CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA4860     GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGA4920     ATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG4980     AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTT5040     CCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA5100     AATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTC5160     TGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGA5220     CAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCG5280     GCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGC5340     GTAAGGAGAAAATACCGCATCAGG5364     (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 6505 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: circular     (ii) MOLECULE TYPE: DNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:     TCGAGGGGGGGCCCGGTACCGATTAGTCCAATTTGTTAAAGACAGGATATCAGGTGGTCC60     AGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGA120     TAGAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTA180     GGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAG240     AATAGAGAAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGG300     ATATCTGTGGTAAGCAGTTCCTGCCCCGCTCAGGGCCAAGAACAGATGGTCCCCAGATGC360     GGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACC420     TGAAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCG480     CGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGT540     CCTCCGATTGACTGCGTCGCCCGGGTACCCGTATTCCCAATAAAGCCTCTTGCTGTTTGC600     ATCCGAATCGTGGACTCGCTGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCAC660     CTCGGGGGTCTTTCATTTGGAGGTTCCACCGAGATTTGGAGACCCCAGCCCAGGGACCAC720     CGACCCCCCCGCCGGGAGGTAAGCTGGCCAGCGGTCGTTTCGTGTCTGTCTCTGTCTTTG780     TGCGTGTTTGTGCCGGCATCTAATGTTTGCGCCTGCGTCTGTACTAGTTGGCTAACTAGA840     TCTGTATCTGGCGGTCCCGCGGAAGAACTGACGAGTTCGTATTCCCGGCCGCAGCCCCTA900     GGAGACGTCCCAGCGGCCTCGGGGGCCCGTTTTGTGGCCCGTTCTGTGTCGTTAACCACC960     CGAGTCGGACTTTTTGGAGCTCCGCCACTGTCCGAGGGGTACGTGGCTTTGTTGGGGGAC1020     GAGAGACAGAGACACTTCCCGCCCCCGTCTGAATTTTTGCTTTCGGTTTTACGCCGAAAC1080     CGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTT1140     CTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCTTAGAT1200     CACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGA1260     TGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGC1320     ACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCAT1380     GGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCT1440     CCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCG1500     TCTCTCCCCCTTGAACCTCCTCTTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTC1560     ACTCCTTCTCTAGGCGGCTCCACCGCGGTGGCGGCCGATCCCCCGGGCTGCAGGAATTCG1620     ATATCAAGCTTATCGATACCGTCGACTCTAGAGGATCCCGCCGCGGCGGGTACCGAGCTC1680     ATTCGAGTAGCGGCTCTTCCAAGCTCAAAGAAGCAGAGGCCGCTGTTCGTTTCCTTTAGG1740     TCTTTCCACTAAAGTCGGAGTATCTTCTTCCAAGATTTCACGTCTTGGTGGCCGTTCCAA1800     GGAGCGCGAGGTCGGGATGGATCTTGAAGGGGACCGCAATGGAGGAGCAAAGAAGAAGAA1860     CTTTTTTAAACTGAACAATAAAAGTGAAAAAGATAAGAAGGAAAAGAAACCAACTGTCAG1920     TGTATTTTCAATGTTTCGCTATTCAAATTGGCTTGACAAGTTGTATATGGTGGTGGGAAC1980     TTTGGCTGCCATCATCCATGGGGCTGGACTTCCTCTCATGATGCTGGTGTTTGGAGAAAT2040     GACAGATATCTTTGCAAATGCAGGAAATTTAGAAGATCTGATGTCAAACATCACTAATAG2100     AAGTGATATCAATGATACAGGGTTCTTCATGAATCTGGAGGAAGACATGACCAGGTATGC2160     CTATTATTACAGTGGAATTGGTGCTGGGGTGCTGGTTGCTGCTTACATTCAGGTTTCATT2220     TTGGTGCCTGGCAGCTGGAAGACAAATACACAAAATTAGAAAACAGTTTTTTCATGCTAT2280     AATGCGACAGGAGATAGGCTGGTTTGATGTGCACGATGTTGGGGAGCTTAACACCCGACT2340     TACAGATGATGTCTCTAAGATTAATGAAGTTATTGGTGACAAAATTGGAATGTTCTTTCA2400     GTCAATGGCAACATTTTTCACTGGGTTTATAGTAGGATTTACACGTGGTTGGAAGCTAAC2460     CCTTGTGATTTTGGCCATCAGTCCTGTTCTTGGACTGTCAGCTGCTGTCTGGGCAAAGAT2520     ACTATCTTCATTTACTGATAAAGAACTCTTAGCGTATGCAAAAGCTGGAGCAGTAGCTGA2580     AGAGGTCTTGGCAGCAATTAGAACTGTGATTGCATTTGGAGGACAAAAGAAAGAACTTGA2640     AAGGTACAACAAAAATTTAGAAGAAGCTAAAAGAATTGGGATAAAGAAAGCTATTACAGC2700     CAATATTTCTATAGGTGCTGCTTTCCTGCTGATCTATGCATCTTATGCTCTGGCCTTCTG2760     GTATGGGACCACCTTGGTCCTCTCAGGGGAATATTCTATTGGACAAGTACTCACTGTATT2820     CTTTTCTGTATTAATTGGGGCTTTTAGTGTTGGACAGGCATCTCCAAGCATTGAAGCATT2880     TGCAAATGCAAGAGGAGCAGCTTATGAAATCTTCAAGATAATTGATAATAAGCCAAGTAT2940     TGACAGCTATTCGAAGAGTGGGCACAAACCAGATAATATTAAGGGAAATTTGGAATTCAG3000     AAATGTTCACTTCAGTTACCCATCTCGAAAAGAAGTTAAGATCTTGAAGGGCCTGAACCT3060     GAAGGTGCAGAGTGGGCAGACGGTGGCCCTGGTTGGAAACAGTGGCTGTGGGAAGAGCAC3120     AACAGTCCAGCTGATGCAGAGGCTCTATGACCCCACAGAGGGGATGGTCAGTGTTGATGG3180     ACAGGATATTAGGACCATAAATGTAAGGTTTCTACGGGAAATCATTGGTGTGGTGAGTCA3240     GGAACCTGTATTGTTTGCCACCACGATAGCTGAAAACATTCGCTATGGCCGTGAAAATGT3300     CACCATGGATGAGATTGAGAAAGCTGTCAAGGAAGCCAATGCCTATGACTTTATCATGAA3360     ACTGCCTCATAAATTTGACACCCTGGTTGGAGAGAGAGGGGCCCAGTTGAGTGGTGGGCA3420     GAAGCAGAGGATCGCCATTGCACGTGCCCTGGTTCGCAACCCCAAGATCCTCCTGCTGGA3480     TGAGGCCACGTCAGCCTTGGACACAGAAAGCGAAGCAGTGGTTCAGGTGGCTCTGGATAA3540     GGCCAGAAAAGGTCGGACCACCATTGTGATAGCTCATCGTTTGTCTACAGTTCGTAATGC3600     TGACGTCATCGCTGGTTTCGATGATGGAGTCATTGTGGAGAAAGGAAATCATGATGAACT3660     CATGAAAGAGAAAGGCATTTACTTCAAACTTGTCACAATGCAGACAGCAGGAAATGAAGT3720     TGAATTAGAAAATGCAGCTGATGAATCCAAAAGTGAAATTGATGCCTTGGAAATGTCTTC3780     AAATGATTCAAGATCCAGTCTAATAAGAAAAAGATCAACTCGTAGGAGTGTCCGTGGATC3840     ACAAGCCCAAGACAGAAAGCTTAGTACCAAAGAGGCTCTGGATGAAAGTATACCTCCAGT3900     TTCCTTTTGGAGGATTATGAAGCTAAATTTAACTGAATGGCCTTATTTTGTTGTTGGTGT3960     ATTTTGTGCCATTATAAATGGAGGCCTGCAACCAGCATTTGCAATAATATTTTCAAAGAT4020     TATAGGGGTTTTTACAAGAATTGATGATCCTGAAACAAAACGACAGAATAGTAACTTGTT4080     TTCACTATTGTTTCTAGCCCTTGGAATTATTTCTTTTATTACATTTTTCCTTCAGGGTTT4140     CACATTTGGCAAAGCTGGAGAGATCCTCACCAAGCGGCTCCGATACATGGTTTTCCGATC4200     CATGCTCAGACAGGATGTGAGTTGGTTTGATGACCCTAAAAACACCACTGGAGCATTGAC4260     TACCAGGCTCGCCAATGATGCTGCTCAAGTTAAAGGGGCTATAGGTTCCAGGCTTGCTGT4320     AATTACCCAGAATATAGCAAATCTTGGGACAGGAATAATTATATCCTTCATCTATGGTTG4380     GCAACTAACACTGTTACTCTTAGCAATTGTACCCATCATTGCAATAGCAGGAGTTGTTGA4440     AATGAAAATGTTGTCTGGACAAGCACTGAAAGATAAGAAAGAACTAGAAGGTGCTGGGAA4500     GATCGCTACTGAAGCAATAGAAAACTTCCGAACCGTTGTTTCTTTGACTCAGGAGCAGAA4560     GTTTGAACATATGTATGCTCAGAGTTTGCAGGTACCATACAGAAACTCTTTGAGGAAAGC4620     ACACATCTTTGGAATTACATTTTCCTTCACCCAGGCAATGATGTATTTTTCCTATGCTGG4680     ATGTTTCCGGTTTGGAGCCTACTTGGTGGCACATAAACTCATGAGCTTTGAGGATGTTCT4740     GTTAGTATTTTCAGCTGTTGTCTTTGGTGCCATGGCCGTGGGGCAAGTCAGTTCATTTGC4800     TCCTGACTATGCCAAAGCCAAAATATCAGCAGCCCACATCATCATGATCATTGAAAAAAC4860     CCCTTTGATTGACAGCTACAGCACGGAAGGCCTAATGCCGAACACATTGGAAGGAAATGT4920     CACATTTGGTGAAGTTGTATTCAACTATCCCACCCGACCGGACATCCCAGTGCTTCAGGG4980     ACTGAGCCTGGAGGTGAAGAAGGGCCAGACGCTGGCTCTGGTGGGCAGCAGTGGCTGTGG5040     GAAGAGCACAGTGGTCCAGCTCCTGGAGCGGTTCTACGACCCCTTGGCAGGGAAAGTGCT5100     GCTTGATGGCAAAGAAATAAAGCGACTGAATGTTCAGTGGCTCCGAGCACACCTGGGCAT5160     CGTGTCCCAGGAGCCCATCCTGTTTGACTGCAGCATTGCTGAGAACATTGCCTATGGAGA5220     CAACAGCCGGGTGGTGTCACAGGAAGAGATCGTGAGGGCAGCAAAGGAGGCCAACATACA5280     TGCCTTCATCGAGTCACTGCCTAATAAATATAGCACTAAAGTAGGAGACAAAGGAACTCA5340     GCTCTCTGGTGGCCAGAAACAACGCATTGCCATAGCTCGTGCCCTTGTTAGACAGCCTCA5400     TATTTTGCTTTTGGATGAAGCCACGTCAGCTCTGGATACAGAAAGTGAAAAGGTTGTCCA5460     AGAAGCCCTGGACAAAGCCAGAGAAGGCCGCACCTGCATTGTGATTGCTCACCGCCTGTC5520     CACCATCCAGAATGCAGACTTAATAGTGGTGTTTCAGAATGGCAGAGTCAAGGAGCATGG5580     CACGCATCAGCAGCTGCTGGCACAGAAAGGCATCTATTTTTCAATGGTCAGTGTCCAGGC5640     TGGAACAAAGCGCCAGTGAACTCTGACTGTATGAGATGTTAAATACTTTTTAATGGGATC5700     CACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTATCCACCATCATGGGGCTTCTCAT5760     TATACTCCTACTCCTACTAATTCTGCTTTTGTGGACCCTGCATTCTTAATCGATTAGTTC5820     AATTTGTTAAAGACAGGATCTCAGTAGTCCAGGCTTTAGTCCTGACTCAACAATACCACC5880     AGCTAAAACCACTAGAATACGAGCCACAATAAATAAAAGATTTTATTTAGTTTCCAGAAA5940     AAGGGGGGAATGAAAGACCCCACCAAGTTGCTTAGCCTGATGCCGCTGTAACGCCATTTT6000     GCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACAT6060     GAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCC6120     GGGGCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGT6180     CCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCC6240     CCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGC6300     TTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGG6360     CGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTTCCCAATAAAGCCTCTT6420     GCTGATTGCATCCGAATCGTGGACTCGCTGATCCTTGGGAGGGTCTCCTCAGATTGATTG6480     ACTGCCCACCTGGGGGTCTTTCAGT6505     (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9318 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: circular     (ii) MOLECULE TYPE: DNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:     TCGAGGGGGGGCCCGGTACCGATTAGTCCAATTTGTTAAAGACAGGATATCAGGTGGTCC60     AGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGA120     TAGAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTA180     GGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAG240     AATAGAGAAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGG300     ATATCTGTGGTAAGCAGTTCCTGCCCCGCTCAGGGCCAAGAACAGATGGTCCCCAGATGC360     GGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACC420     TGAAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCG480     CGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGT540     CCTCCGATTGACTGCGTCGCCCGGGTACCCGTATTCCCAATAAAGCCTCTTGCTGTTTGC600     ATCCGAATCGTGGACTCGCTGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCAC660     CTCGGGGGTCTTTCATTTGGAGGTTCCACCGAGATTTGGAGACCCCAGCCCAGGGACCAC720     CGACCCCCCCGCCGGGAGGTAAGCTGGCCAGCGGTCGTTTCGTGTCTGTCTCTGTCTTTG780     TGCGTGTTTGTGCCGGCATCTAATGTTTGCGCCTGCGTCTGTACTAGTTGGCTAACTAGA840     TCTGTATCTGGCGGTCCCGCGGAAGAACTGACGAGTTCGTATTCCCGGCCGCAGCCCCTA900     GGAGACGTCCCAGCGGCCTCGGGGGCCCGTTTTGTGGCCCGTTCTGTGTCGTTAACCACC960     CGAGTCGGACTTTTTGGAGCTCCGCCACTGTCCGAGGGGTACGTGGCTTTGTTGGGGGAC1020     GAGAGACAGAGACACTTCCCGCCCCCGTCTGAATTTTTGCTTTCGGTTTTACGCCGAAAC1080     CGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTT1140     CTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCTTAGAT1200     CACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGA1260     TGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGC1320     ACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCAT1380     GGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCT1440     CCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCG1500     TCTCTCCCCCTTGAACCTCCTCTTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTC1560     ACTCCTTCTCTAGGCGGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGGATCCCGC1620     CGCGGCGGGTACCGAGCTCATTCGAGTAGCGGCTCTTCCAAGCTCAAAGAAGCAGAGGCC1680     GCTGTTCGTTTCCTTTAGGTCTTTCCACTAAAGTCGGAGTATCTTCTTCCAAGATTTCAC1740     GTCTTGGTGGCCGTTCCAAGGAGCGCGAGGTCGGGATGGATCTTGAAGGGGACCGCAATG1800     GAGGAGCAAAGAAGAAGAACTTTTTTAAACTGAACAATAAAAGTGAAAAAGATAAGAAGG1860     AAAAGAAACCAACTGTCAGTGTATTTTCAATGTTTCGCTATTCAAATTGGCTTGACAAGT1920     TGTATATGGTGGTGGGAACTTTGGCTGCCATCATCCATGGGGCTGGACTTCCTCTCATGA1980     TGCTGGTGTTTGGAGAAATGACAGATATCTTTGCAAATGCAGGAAATTTAGAAGATCTGA2040     TGTCAAACATCACTAATAGAAGTGATATCAATGATACAGGGTTCTTCATGAATCTGGAGG2100     AAGACATGACCAGGTATGCCTATTATTACAGTGGAATTGGTGCTGGGGTGCTGGTTGCTG2160     CTTACATTCAGGTTTCATTTTGGTGCCTGGCAGCTGGAAGACAAATACACAAAATTAGAA2220     AACAGTTTTTTCATGCTATAATGCGACAGGAGATAGGCTGGTTTGATGTGCACGATGTTG2280     GGGAGCTTAACACCCGACTTACAGATGATGTCTCTAAGATTAATGAAGTTATTGGTGACA2340     AAATTGGAATGTTCTTTCAGTCAATGGCAACATTTTTCACTGGGTTTATAGTAGGATTTA2400     CACGTGGTTGGAAGCTAACCCTTGTGATTTTGGCCATCAGTCCTGTTCTTGGACTGTCAG2460     CTGCTGTCTGGGCAAAGATACTATCTTCATTTACTGATAAAGAACTCTTAGCGTATGCAA2520     AAGCTGGAGCAGTAGCTGAAGAGGTCTTGGCAGCAATTAGAACTGTGATTGCATTTGGAG2580     GACAAAAGAAAGAACTTGAAAGGTACAACAAAAATTTAGAAGAAGCTAAAAGAATTGGGA2640     TAAAGAAAGCTATTACAGCCAATATTTCTATAGGTGCTGCTTTCCTGCTGATCTATGCAT2700     CTTATGCTCTGGCCTTCTGGTATGGGACCACCTTGGTCCTCTCAGGGGAATATTCTATTG2760     GACAAGTACTCACTGTATTCTTTTCTGTATTAATTGGGGCTTTTAGTGTTGGACAGGCAT2820     CTCCAAGCATTGAAGCATTTGCAAATGCAAGAGGAGCAGCTTATGAAATCTTCAAGATAA2880     TTGATAATAAGCCAAGTATTGACAGCTATTCGAAGAGTGGGCACAAACCAGATAATATTA2940     AGGGAAATTTGGAATTCAGAAATGTTCACTTCAGTTACCCATCTCGAAAAGAAGTTAAGA3000     TCTTGAAGGGCCTGAACCTGAAGGTGCAGAGTGGGCAGACGGTGGCCCTGGTTGGAAACA3060     GTGGCTGTGGGAAGAGCACAACAGTCCAGCTGATGCAGAGGCTCTATGACCCCACAGAGG3120     GGATGGTCAGTGTTGATGGACAGGATATTAGGACCATAAATGTAAGGTTTCTACGGGAAA3180     TCATTGGTGTGGTGAGTCAGGAACCTGTATTGTTTGCCACCACGATAGCTGAAAACATTC3240     GCTATGGCCGTGAAAATGTCACCATGGATGAGATTGAGAAAGCTGTCAAGGAAGCCAATG3300     CCTATGACTTTATCATGAAACTGCCTCATAAATTTGACACCCTGGTTGGAGAGAGAGGGG3360     CCCAGTTGAGTGGTGGGCAGAAGCAGAGGATCGCCATTGCACGTGCCCTGGTTCGCAACC3420     CCAAGATCCTCCTGCTGGATGAGGCCACGTCAGCCTTGGACACAGAAAGCGAAGCAGTGG3480     TTCAGGTGGCTCTGGATAAGGCCAGAAAAGGTCGGACCACCATTGTGATAGCTCATCGTT3540     TGTCTACAGTTCGTAATGCTGACGTCATCGCTGGTTTCGATGATGGAGTCATTGTGGAGA3600     AAGGAAATCATGATGAACTCATGAAAGAGAAAGGCATTTACTTCAAACTTGTCACAATGC3660     AGACAGCAGGAAATGAAGTTGAATTAGAAAATGCAGCTGATGAATCCAAAAGTGAAATTG3720     ATGCCTTGGAAATGTCTTCAAATGATTCAAGATCCAGTCTAATAAGAAAAAGATCAACTC3780     GTAGGAGTGTCCGTGGATCACAAGCCCAAGACAGAAAGCTTAGTACCAAAGAGGCTCTGG3840     ATGAAAGTATACCTCCAGTTTCCTTTTGGAGGATTATGAAGCTAAATTTAACTGAATGGC3900     CTTATTTTGTTGTTGGTGTATTTTGTGCCATTATAAATGGAGGCCTGCAACCAGCATTTG3960     CAATAATATTTTCAAAGATTATAGGGGTTTTTACAAGAATTGATGATCCTGAAACAAAAC4020     GACAGAATAGTAACTTGTTTTCACTATTGTTTCTAGCCCTTGGAATTATTTCTTTTATTA4080     CATTTTTCCTTCAGGGTTTCACATTTGGCAAAGCTGGAGAGATCCTCACCAAGCGGCTCC4140     GATACATGGTTTTCCGATCCATGCTCAGACAGGATGTGAGTTGGTTTGATGACCCTAAAA4200     ACACCACTGGAGCATTGACTACCAGGCTCGCCAATGATGCTGCTCAAGTTAAAGGGGCTA4260     TAGGTTCCAGGCTTGCTGTAATTACCCAGAATATAGCAAATCTTGGGACAGGAATAATTA4320     TATCCTTCATCTATGGTTGGCAACTAACACTGTTACTCTTAGCAATTGTACCCATCATTG4380     CAATAGCAGGAGTTGTTGAAATGAAAATGTTGTCTGGACAAGCACTGAAAGATAAGAAAG4440     AACTAGAAGGTGCTGGGAAGATCGCTACTGAAGCAATAGAAAACTTCCGAACCGTTGTTT4500     CTTTGACTCAGGAGCAGAAGTTTGAACATATGTATGCTCAGAGTTTGCAGGTACCATACA4560     GAAACTCTTTGAGGAAAGCACACATCTTTGGAATTACATTTTCCTTCACCCAGGCAATGA4620     TGTATTTTTCCTATGCTGGATGTTTCCGGTTTGGAGCCTACTTGGTGGCACATAAACTCA4680     TGAGCTTTGAGGATGTTCTGTTAGTATTTTCAGCTGTTGTCTTTGGTGCCATGGCCGTGG4740     GGCAAGTCAGTTCATTTGCTCCTGACTATGCCAAAGCCAAAATATCAGCAGCCCACATCA4800     TCATGATCATTGAAAAAACCCCTTTGATTGACAGCTACAGCACGGAAGGCCTAATGCCGA4860     ACACATTGGAAGGAAATGTCACATTTGGTGAAGTTGTATTCAACTATCCCACCCGACCGG4920     ACATCCCAGTGCTTCAGGGACTGAGCCTGGAGGTGAAGAAGGGCCAGACGCTGGCTCTGG4980     TGGGCAGCAGTGGCTGTGGGAAGAGCACAGTGGTCCAGCTCCTGGAGCGGTTCTACGACC5040     CCTTGGCAGGGAAAGTGCTGCTTGATGGCAAAGAAATAAAGCGACTGAATGTTCAGTGGC5100     TCCGAGCACACCTGGGCATCGTGTCCCAGGAGCCCATCCTGTTTGACTGCAGCATTGCTG5160     AGAACATTGCCTATGGAGACAACAGCCGGGTGGTGTCACAGGAAGAGATCGTGAGGGCAG5220     CAAAGGAGGCCAACATACATGCCTTCATCGAGTCACTGCCTAATAAATATAGCACTAAAG5280     TAGGAGACAAAGGAACTCAGCTCTCTGGTGGCCAGAAACAACGCATTGCCATAGCTCGTG5340     CCCTTGTTAGACAGCCTCATATTTTGCTTTTGGATGAAGCCACGTCAGCTCTGGATACAG5400     AAAGTGAAAAGGTTGTCCAAGAAGCCCTGGACAAAGCCAGAGAAGGCCGCACCTGCATTG5460     TGATTGCTCACCGCCTGTCCACCATCCAGAATGCAGACTTAATAGTGGTGTTTCAGAATG5520     GCAGAGTCAAGGAGCATGGCACGCATCAGCAGCTGCTGGCACAGAAAGGCATCTATTTTT5580     CAATGGTCAGTGTCCAGGCTGGAACAAAGCGCCAGTGAACTCTGACTGTATGAGATGTTA5640     AATACTTTTTAATGGGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACC5700     GTCGACCTCGAGGGGGGGCCCGGTACAGATTAGTCCAATTTGTTAAAGACAGGATATCAG5760     GTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAG5820     CCATAGATAGAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCC5880     ACCTGTAGGTTTGGCAAGGCTAGCTTAAGTAAGCCATTTTGCAAGGCATGGAAAAATACA5940     TAACTGAGAATAGAGAAGTTCAGATCAAGGTTAGGAACAGAGAGACAGGAGAATATGGGC6000     CAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAA6060     CAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGC6120     CAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGA6180     TGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATC6240     AGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCAC6300     AACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTATTCC6360     CAATAAAGCCTCTTGCTGTTTGCATCCGAATCGTGGACTCGCTGATCCTTGGGAGGGTCT6420     CCTCAGATTGATTGACTGCCCACCTCGGGGGTCTTTCAGTAGGATCTCGACCGATGCCCT6480     TGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCG6540     CACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGG6600     TCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGG6660     TATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTT6720     TCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGC6780     TGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCG6840     GCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGG6900     GACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCACTGGACCGCTGA6960     TCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAG7020     GCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTG7080     CACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAAC7140     ACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGT7200     GACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAG7260     ACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTC7320     TTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTT7380     CTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATA7440     ATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTT7500     TGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGC7560     TGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT7620     CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCT7680     ATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACA7740     CTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGG7800     CATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAA7860     CTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGG7920     GGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGA7980     CGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGG8040     CGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGT8100     TGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGG8160     AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTC8220     CCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA8280     GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTC8340     ATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGAT8400     CCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTC8460     AGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTG8520     CTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT8580     ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCT8640     TCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCT8700     CGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGG8760     GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTC8820     GTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA8880     GCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG8940     CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTA9000     TAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGG9060     GGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG9120     CTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTAT9180     TACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTC9240     AGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC9300     GATTCATTAATGCAGNNG9318     __________________________________________________________________________ 

We claim:
 1. A retroviral vector hybrid comprisinga) a 5'-LTR-comprising at least one U5 region or tRNA primer binding site wherein the region or site is selected from the group consisting of the U5 region of MESV, the tRNA primer binding site of MESV, the U5 region of MoMuSV, and the tRNA primer binding site of MoMuSV, and b) a 3'-LTR comprising the U3 and R regions from a Friend murine leukaemia virus (F-MuLV).
 2. The vector hybrid of claim 1 further comprising the leader region of MESV.
 3. The vector hybrid of claim 1, wherein the Friend murine leukaemia virus is selected from the group consisting of malignant histiosarcoma virus (MHSV), SFFVp Lilly-Steeves, SFFVa, Rauscher SSFV, F-muLVc157 and Friend-mink cell focus forming Virus.
 4. The vector hybrid of claim 1, wherein the U3 and R regions in the 5'-LTR comprise the U3 and R regions from MPSV, MESV or PCMV.
 5. The vector hybrid of claim 1, wherein the 5'-LTR comprises bp 142-657 from SEQ ID NO:1.
 6. The vector hybrid of claim 1, wherein the 3'-LTR comprises bp 1707-2283 from SEQ ID NO:1.
 7. The vector hybrid of claim 1 further comprising at least one gene that is heterologous to the retroviral vector and can be expressed in eukaryotic cells.
 8. The vector hybrid of claim 1 which is replication-defective.
 9. The vector hybrid of claim 1 which is replication-competent.
 10. The vector hybrid of claim 7 wherein the heterologous gene is selected from the group consisting of a multiple drug resistance gene (MDR gene), an antibiotic resistance gene, a LNGFR gene, a cerebrosidase gene and the herpes simplex TK gene.
 11. The vector of claim 10 wherein the heterologous gene is the neo^(R) gene.
 12. A vector hybrid selected from the group consisting of pSF1, pSF2, pSF3, and pHM1.
 13. A process for the production of a retrovirally transduced eukaryotic cell which expresses an exogenous gene, said process comprising transducing a eukaryotic cell with a retroviral vector virus, which virus comprisesa) at least one U5 region or tRNA primer binding site wherein the region or site is selected from the group consisting of the U5 region of MESV, the tRNA primer binding site of MESV, the U5 region of MoMuSV, and the tRNA primer binding site of MoMuSV, and b) a 3'-LTR comprising the U3 and R regions from a Friend murine leukaemia virus (F-MuLV).
 14. The process as claimed in claim 13 wherein the retroviral vector virus is replication defective.
 15. The process as claimed in claim 13 wherein the eukaryotic cells is a haematopoietic stem cells.
 16. An eukaryotic cell obtained by the process of transducing a eukaryotic cell with a retroviral vector virus comprisinga) at least one U5 region or tRNA primer binding site wherein the region or site is selected from the group consisting of the U5 region of MESV, the tRNA primer binding site of MESV, the U5 region of MoMuSV, and the tRNA primer binding site of MoMuSV, and b) a 3'-LTR comprising the U3 and R regions from a Friend murine leukaemia virus (F-MuLV).
 17. The cell of claim 16 wherein the F-MuLV is selected from the group consisting of malignant histiosarcoma virus (MHSV), SFFVp Lilly-Steeves, SFFVa, Rauscher SSFV, F-muLVc157 and Friend-mink cell focus forming virus.
 18. The cell of claim 16 wherein the vector further comprises the leader region of MESV.
 19. The cell of claim 16 wherein the retroviral vector virus is replication defective.
 20. A replication defective infectious virus particle comprising a retroviral RNA genome, wherein the genome comprisesa) at least one U5 region or tRNA primer binding site selected from the group consisting of the U5 region of MESV, the tRNA primer binding site of MESV, the U5 region of MoMuSV, and the tRNA primer binding site of MoMuSV, and b) further comprises a packaging function, a gene which is heterologous to the virus and can be expressed in the eukaryotic cell, and U3 and R from a Friend murine leukaemia virus (F-MuLV), but c) does not comprise active gag, env and pol sequences at the 3' end.
 21. A process for the production of a replication defective infectious virus particle comprisinga) transfecting a eukaryotic helper cell which has the helper functions gag, env and pol with a vector hybrid comprisingi) at least one U5 region or tRNA primer binding site wherein the region or site is selected from the group consisting of the U5 region of MESV, the tRNA primer binding site of MESV, the U5 region of MoMuSV, and the tRNA primer binding site of MoMuSV, and ii) a 3'-LTR comprising the U3 and R regions from a Friend murine leukaemia virus (F-MuLV), b) producing the RNA corresponding to the DNA of the vector hybrid as the virus genome in the cell, c) packaging the virus genome into the replication deficient empty virus envelopes formed in the cell, and d) isolating the infectious virus particles which contain said virus genome.
 22. The process of claim 21 wherein the F-MuLV is selected from the group consisting of malignant histiosarcoma virus (MHSV), SFFVp Lilly-Steeves, SFFVa, Rauscher SFFV, F-muLVc 157 and Friend-mink cell focus forming virus.
 23. The process of claim 21 wherein the vector hybrid further comprises the leader region of MESV.
 24. A retroviral vector hybrid comprisinga) the U5 region and tRNA primer binding site of MESV as the U5 region and the tRNA primer binding site in the leader region, and b) the U3 and R regions from myeloproliferative sarcoma virus (MPSV) as the U3 and R regions in the 3'-LTR.
 25. A process for the production of a retrovirally transduced eukaryotic cell comprising an exogenous gene, wherein the eukaryotic cell is transduced with the retroviral vector virus comprising:a) the U5 region and/or tRNA primer binding site of MESV as the U5 region and/or tRNA primer binding site in the leader region, b) the U3 and R regions from myeloproliferative sarcoma virus (MPSV) as the U3 and R regions in the 3'-LTR and c) the said exogenous gene.
 26. A replication defective infectious virus particle comprising retroviral RNA as the genome, wherein the genome comprises the U5 regions and/or tRNA primer binding site of MESV as the U5 region and/or tRNA primer binding site, a packaging function and a gene which is heterologous for the virus and can be expressed in the eukaryotic cell, and U3 and R from the myeloproliferative sarcoma virus (MPSV) but no active gag, env and pol sequences at the 3' end. 